Human embryonic stem cells (hESCs) have strong potential as sources of cells for the treatment for disease and injury (e.g. tissue engineering and reconstruction, diabetes, Parkinson's Disease, leukemia, congestive heart failure, etc.). The successful integration of hESC into such therapies will hinge upon three critical steps: their expansion without differentiation (i.e., self-renewal), their differentiation into a specific cell type or collection of cell types, and the promotion of their survival and functional integration into existing tissue. However, controlling cell behavior during each of these steps will require precise control over the cellular microenvironment. This poses a major challenge ex vivo in current hESC culture systems, which range from co-culture with feeder cells to serum-free systems where cells are cultured on complex extracellular matrix proteins. All such systems involve animal or human proteins, which pose problems for pathogen transmission, immune rejection, limited reproducibility, and scale up to a clinical process. To achieve the intended goals of regenerative medicine, methods for the precise control of the survival, proliferation, and differentiation of stem cell populations in vitro and in vivo are necessary. Here, we propose to develop a completely synthetic environment to precisely control hESC self-renewal in culture. Specifically, we will engineer a tunable and well-defined environment presenting a completely "synthetic extracellular matrix" (ECM) and chemically-defined media to control the self-renewal/expansion of hESCs. Furthermore, we will develop high throughput approaches to identify synthetic peptide ligands for functionalization to the synthetic ECM and promotion of hESC self-renewal. If hESCs can be derived and maintained within this fully synthetic microenvironment, then it will be possible to eliminate pathogen transmission associated with mouse or human feeder layers, provide a scalable basis for large-scale production of hESCs, and provide a precise base for further development to control hES cell differentiation. Furthermore, the result will be a technology platform that can be generally applied to numerous stem cell populations and used to investigate the basic biological/developmental mechanisms underlying self-renewal. Public Health Relevance: The development of novel, bioactive materials has significant potential for exerting precise control over cell function, both for fundamental biological studies and applications in tissue engineering and regenerative medicine. For example, developing synthetic, bioactive material systems to promote the self-renewal and expansion of human embryonic stem cells will have numerous biomedical applications including the design of therapies for disease or injury in the muscle, bone, brain, heart, liver, pancreas, and other tissues. The novel blend of stem cell biology, materials science, molecular biology, and bioengineering described in this proposal will be well suited to addressing an important problem, i.e. stem cell control, at the interface of biology, engineering, and medicine